A safe and effective HIV vaccine amicable for use in most parts of the world to prevent and/or treat HIV infection through strong mucosal and systemic immunity is an urgent need. This innovation grant proposal will test the potential of a highly conserved HIV envelope peptide cocktail we developed along with a novel cholera toxin mutant as adjuvant for mucosal vaccination, preferably by the oral route. Previous studies demonstrated that prophylactic vaccination with this peptide cocktail delivered by intravenous route using Freund's adjuvant and/or autologous dendritic cells protected rhesus macaques from chronic infection and AIDS by SHIVku2 and SHIV89.6P. A two-codon mutant of cholera toxin, CT2*, we developed (patent pending), was effective as adjuvant for intranasal immunization of mice with the HIV peptides for inducing HIV-specific mucosal and systemic cellular immune responses. We also observed that oral immunization of mice with a CTL-epitope peptide and CT2*, after neutralizing stomach acid with bicarbonate, resulted in induction of antigen-specific CTL responses. We hypothesize that the highly conserved HIV envelope peptide cocktail when combined with CT2* will be an efficient vaccine for mucosal immunization by intranasal as well as oral delivery. To test this hypothesis, we will first confirm whether the two-codon mutant of cholera toxin CT2* we developed will be superior to another well-established single-codon mutant E112K in terms of adjuvanticity. After this, we propose comparative analysis in the mouse model, for delivery of peptide cocktail along with CT2* by the intranasal and oral routes to generate antigen-specific cellular immune responses that will be assessed for protective efficacy against challenge with recombinant vaccinia virus encoding the HIV envelope protein. For oral immunization, we also propose to test a strategy involving encapsulation of the vaccine peptide cocktail in enteric-coated capsules that are designed to dissolve when encountered with acid in the digestive track and deliver the vaccine to intestinal antigen presenting cells (in collaboration with Dr. Michael Barry, Baylor college of Medicine). The optimal mucosal delivery strategy (intranasal or oral) selected from the mouse studies will be used for testing immunogenicity and protective efficacy of the peptide cocktail vaccine admixed with the CT2* adjuvant in rhesus macaques against intra-vaginal pathogenic SHIV challenge. Successful outcome in these studies should enable us to formulate pre-clinical development plan for mucosal vaccination with the peptide cocktail. [unreadable] [unreadable] [unreadable]